Engineered Chimeric Protein Antigens for Porcine Diseases

According to previous studies of Mhp and PCV2, coding DNA sequences (CDS) of P97R1 (MHP168_110), P46 (MHP168_522), and P42 (MHP168_069) from Mhp strain 168 (GenBank CP002274.1) were used to construct the chimeric protein antigen; Cap protein (deleting the nuclear localization signal of 41 amino acid residues) from PCV2 isolate strain ShanDong3-2016 (KY656098.1) was used as another antigen (Table 1) [9 (link),15 (link),32 (link)]. EGFP and mCherry were used as tag proteins for detection and were cloned from vectors pEGFP-N1 and pmCherry-N1 (Clontech, Otsu, Shiga, Japan), respectively. SPgp64 (GenBank AFO10080.1) and TMDgp64 (GenBank CAA24524.1) were located upstream of the target protein and downstream of the tag protein, respectively.
Flexible linker GGSG (GlyGlySerGly) was inserted between each chimeric subunit protein of P97R1P46P42 to achieve proper folding. The fusion gene fragment SPgp64-P97R1P46P42-mCherry-TMDgp64-6×His was inserted into the baculovirus expression vector pFastBac dual plasmid (Invitrogen, Carlsbad, CA, USA) to generate pFastBac dual-P97R1P46P42-mCherry-Cap-EGFP. SPgp64-Cap-EGFP-TMDgp64-6×His was inserted into pFastBac dual to generate pFastBac dual-P97R1P46P42-Cap.